Antisense nucleic acid targeting pcsk9

ABSTRACT

Provided is an oligonucleotide conjugate comprising an oligonucleotide and two or more linearly connected asialoglycoprotein receptor-binding molecules attached to the oligonucleotide, wherein the oligonucleotide comprises a locked nucleoside analog having a bridging structure between the 4′ and 2′ positions, is complementary to a human PCSK9 gene, and has inhibitory activity on the expression of the human PCSK9 gene. The oligonucleotide conjugate of the present invention can be used in the field of pharmaceutical products, in particular, the field of the development and production of therapeutic agents for diseases associated with a high LDL cholesterol level.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of and claims the benefit andpriority to U.S. patent application Ser. No. 16/616,724, filed on Nov.25, 2019, which is a U.S. National Phase Application of PCTInternational Application Number PCT/JP2018/020081, filed on May 24,2018, designating the United States of America and published in theJapanese language, which is an International Application of and claimsthe benefit of priority to Japanese Patent Application No. 2017-105121,filed on May 26, 2017. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSegList-IWAT015-002C1.txt, the date of creation of the ASCII text fileis Feb. 2, 2022, and the size of the ASCII text file is 18 KB.

TECHNICAL FIELD

The present invention relates to an antisense nucleic acid targetingPCSK9 and a pharmaceutical composition comprising the same.

BACKGROUND ART

Familial hypercholesterolemia (FH) is a condition characterized byhyper-LDL-cholesterolemia, which causes early onset and progression ofatherosclerosis, often leading to the development of arterioscleroticdiseases such as myocardial infarction. For the treatment of FH, forexample, lipid-lowering agents such as statins are used to controlLDL-C.

Statins inhibit cholesterol synthesis, resulting in increased LDLreceptor activity and LDL-C level reduction; however statins upregulatethe expression of PCSK9, which is responsible for LDL receptordegradation. For this reason, standard therapy using statins orezetimibe is not sufficiently effective for severe hypercholesterolemiasuch as FH and cannot prevent the progression of atherosclerosis, andthus there are not a few cases in which cardiovascular events repeatedlyoccur even during therapy. To patients with severe hypercholesterolemiaor a type of FH refractory to medication alone, a therapy called LDLapheresis has been provided. However, this therapy imposes heavyphysical and temporal burden to the patients.

A study showed that, in carriers of a loss-of-function PCSK9 variant, alower LDL-C level and an 88% lower incidence rate of coronary arterydisease were observed as compared with those in noncarriers (see NonPatent Literature 1). This finding indicates that PCSK9 is a promisingtarget molecule of dyslipidemia. However, PCSK9 has no active center,which is an obstacle to the development of small molecule compoundsserving as PCSK9 inhibitors. Under such circumstances, a novel form ofPCSK9 synthesis inhibitor, namely, a PCSK9 antisense nucleic acid, isbeing actively researched rather than inhibitors directly acting onPCSK9.

For example, Patent Literature 1 discloses an about 10- to 30-merantisense oligonucleotide against the human PCSK9 gene, which antisenseoligonucleotide is capable of inhibiting the expression of PCSK9. Thisantisense oligonucleotide can be used in the form of a conjugate inwhich a protein, a fatty acid chain, a sugar residue, or the like, oreven a drug substance such as an antibiotic is attached to the antisenseoligonucleotide.

Patent Literature 2 discloses a 10- to 22-mer antisense oligonucleotidewhich is capable of inhibiting the activity of PCSK9 and lessnephrotoxic. Also disclosed are embodiments in which a conjugate isformed from the antisense oligonucleotide and a sterol such ascholesterol or a carbohydrate such as N-acetylgalactosamine (GalNAc). Inparticular, tri-antennary GalNAc-conjugated antisense oligonucleotidesare preferred in terms of binding affinity for the liver.

CITATION LIST Patent Literature

-   Patent Literature 1:

U.S. patent application publication No. 2012/0122954

-   Patent Literature 2:

WO 2014/207232

Non Patent Literature

-   Non Patent Literature 1:

Cohen J C, et al., N Engl J Med 2006, 354, 1264-1272.

SUMMARY OF INVENTION Technical Problem

In some cases, antisense nucleic acids may form higher-order structuresor complexes, which affect the pharmacokinetics and efficacy of theantisense nucleic acids. Therefore, in order to enhance the activity ofantisense nucleic acids in the body, pharmacokinetic properties of theantisense nucleic acids have to be taken into consideration.

In addition, although antisense nucleic acids are potentially usefulpharmaceutical materials, those with low activity and poor tissuespecificity may cause unexpected adverse effects. In fact, SPC5001,which had been under development as an antisense nucleic acid inhibitorof PCSK9, caused damage to the kidney, which was a non-target tissue, inthe phase I trial conducted on healthy volunteers, and for this reason,its development was terminated (see van Poelgeest E P, et al., Am JKidney Dis 2013, 62, 796-800; and van Poelgeest E P, et al., Br J ClinPharmacol 2015, 80, 1350-1361). Therefore, for clinical application ofantisense nucleic acids targeting PCSK9, there is a need for improvementof their tissue specificity.

The present invention provides an antisense nucleic acid which targetsPCSK9 and has in vivo efficacy and low toxicity and also provides apharmaceutical composition comprising the same.

Solution to Problem

Generally, the activity of an antisense nucleic acid is evaluated fromthe measurement of target mRNA reduction by the antisense nucleic acidintroduced into cells with a cationic lipid-based reagent, such asLipofectamine (by lipofection). However, this method cannot alwaysevaluate the precise activity of the antisense nucleic acid. One reasonis that the efficiency of complexation of the reagent with the antisensenucleic acid may vary with its sequence. Another reason is that thismethod may abolish pharmacokinetic properties inherent to the sequenceof the antisense nucleic acid.

The present inventors conducted extensive research and found thatantisense nucleic acids of PCSK9 which are efficacious in vivo and lesstoxic can be prepared by designing nucleic acid sequences with highbiostability, selecting those with high binding affinity for a PCSK9gene using a technique called the Ca²⁺ enrichment of medium (CEM)method, and modifying the selected nucleic acid sequences with aspecific sugar residue. Based on this finding, the present inventorscompleted the present invention.

That is, the present invention relates to the following. [1] Anoligonucleotide conjugate comprising an oligonucleotide and two or morelinearly connected asialoglycoprotein receptor-binding moleculesattached to the oligonucleotide, wherein the oligonucleotide comprises alocked nucleoside analog having a bridging structure between the 4′ and2′ positions, is complementary to a human PCSK9 gene, and has inhibitoryactivity on expression of the human PCSK9 gene.

[2] The oligonucleotide conjugate according to the above [1], whereinthe bridging structure is selected from the following (i) to (iv):(i) a structure represented by —CH₂—O— or —(CH₂)₂—O—;(ii) a structure represented by —CH₂—NR¹—O— or —(CH₂)₂—NR¹—O—;(iii) a structure represented by —CO—NR¹—, —CH₂—CO—NR¹—,—(CH₂)₂—CO—NR¹—, —CO—NR¹—X—, or —CH₂—CO—NR¹—X—; and(iv) a structure represented by —CH₂—NR¹— or —(CH₂)₂—NR¹—(wherein R¹represents a hydrogen atom;an optionally branched or cyclic alkyl group of 1 to 7 carbon atoms;an optionally branched or cyclic alkenyl group of 2 to 7 carbon atoms;an aryl group of 3 to 12 carbon atoms which may have a heteroatom andmay have any one or more substituting groups selected from group αconsisting of a hydroxyl group, a straight-chain alkyl group of 1 to 6carbon atoms, a straight-chain alkoxy group of 1 to 6 carbon atoms, amercapto group, a straight-chain alkylthio group of 1 to 6 carbon atoms,an amino group, a straight-chain alkylamino group of 1 to 6 carbonatoms, and a halogen atom; or an alkyl group having an aryl moiety of 3to 12 carbon atoms which moiety may have a heteroatom and may have anyone or more substituting groups selected from the group α, andX represents an oxygen atom, a sulfur atom, an amino group, or amethylene group).[3] The oligonucleotide conjugate according to the above [1] or [2],wherein the human PCSK9 gene is a region represented by a nucleotidesequence comprising any of the following: the nucleotide sequence of SEQID NO: 3; the nucleotide sequence of SEQ ID NO: 4; the nucleotidesequence of SEQ ID NO: 5; the nucleotide sequence of SEQ ID NO: 6; thenucleotide sequence of SEQ ID NO: 7; the nucleotide sequence of SEQ IDNO: 8; the nucleotide sequence of SEQ ID NO: 9; the nucleotide sequenceof SEQ ID NO: 10; the nucleotide sequence of SEQ ID NO: 11; thenucleotide sequence of SEQ ID NO: 12; the nucleotide sequence of SEQ IDNO: 13; the nucleotide sequence of SEQ ID NO: 14; and complementarynucleotide sequences thereof.[4] The oligonucleotide conjugate according to any of the above [1] to[3], wherein one or more internucleoside linkages are phosphorothioatelinkages.[5] The oligonucleotide conjugate according to any of the above [1] to[4], wherein one or more linkages selected from a linkage between theasialoglycoprotein receptor-binding molecules and a linkage between theoligonucleotide and the asialoglycoprotein receptor-binding moleculesare phosphodiester linkages.[6] The oligonucleotide conjugate according to any of the above [1] to[5], wherein the linkage between the oligonucleotide and theasialoglycoprotein receptor-binding molecules is a linkage via a linkerselected from the following (A) and (B).

[7] The oligonucleotide conjugate according to any of the above [1] to[6], wherein the number of the asialoglycoprotein receptor-bindingmolecules linearly connected is 2 to 5.[8] The oligonucleotide conjugate according to any of the above [1] to[7], wherein the asialoglycoprotein receptor-binding molecules are oneor more types of molecules selected from the group consisting oflactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine,N-formylgalactosamine, N-propionylgalactosamine,N-n-butanoylgalactosamine, N-iso-butanoylgalactosamine, and derivativesthereof.[9] The oligonucleotide conjugate according to any of the above [1] to[8], wherein the oligonucleotide has a 10- to 25-base nucleotidesequence.[10] A preventive or therapeutic agent for a disease associated with ahigh LDL cholesterol level, the preventive or therapeutic agentcomprising the oligonucleotide conjugate according to any of the above[1] to [9] as an active ingredient.[11] The preventive or therapeutic agent according to the above [10],wherein the disease associated with a high LDL cholesterol level isselected from hypercholesterolemia and high-risk diseases in more needof LDL cholesterol reduction.[12] The preventive or therapeutic agent according to the above [10] or[11], wherein the preventive or therapeutic agent is an injectablepreparation.

Advantageous Effects of Invention

The antisense nucleic acid of the present invention has great advantagesin that it is capable of inhibiting PCSK9 expression in vivo and is lesstoxic, and thus is useful as a therapeutic agent for a diseaseassociated with a high LDL cholesterol level.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of primary screening for antisense activityusing the CEM method.

FIG. 2 shows the results of the examination of the concentrationdependency of the activity of the antisense nucleic acids selected inthe primary screening.

FIG. 3 shows the change in blood LDL cholesterol level in cynomolgusmonkeys in conjunction with the dose of the antisense nucleic acid(relative value compared to the level at the start of the test).

FIG. 4 shows the change in blood PCSK9 level in cynomolgus monkeys afterantisense nucleic acid administration at 10 mg/kg (relative valuecompared to the level at the time point of 10 mg/kg administration).

FIG. 5 shows the appearance of the kidneys of cynomolgus monkeys afterintermittent administration of an antisense nucleic acid at 10 mg/kg or30 mg/kg.

FIG. 6 shows the urine protein level in cynomolgus monkeys afterintermittent administration of an antisense nucleic acid at 10 mg/kg or30 mg/kg.

FIG. 7 shows the blood urea nitrogen level in cynomolgus monkeys afterintermittent administration of an antisense nucleic acid at 10 mg/kg or30 mg/kg.

FIG. 8 shows the blood creatinine level in cynomolgus monkeys afterintermittent administration of an antisense nucleic acid at 10 mg/kg or30 mg/kg.

FIG. 9 shows the blood urea nitrogen level in rats after intermittentadministration of an antisense nucleic acid at 10 mg/kg or 30 mg/kg.

FIG. 10 shows the blood creatinine level in rats after intermittentadministration of an antisense nucleic acid at 10 mg/kg or 30 mg/kg.

FIG. 11 is a schematic view of the structure of an antisense nucleicacid-GalNAc conjugate.

FIG. 12 shows the change in blood LDL cholesterol level in cynomolgusmonkeys after single administration of an antisense nucleic acid-GalNAcconjugate at 0.3 mg/kg or 1 mg/kg (relative value compared to the levelat the start of the test).

FIG. 13 shows the appearance of the kidneys of rats after intermittentadministration of an antisense nucleic acid or an antisense nucleicacid-GalNAc conjugate.

FIG. 14 shows the urinary output in rats after intermittentadministration of an antisense nucleic acid or an antisense nucleicacid-GalNAc conjugate.

FIG. 15 shows the urine Kim-1 level in rats after intermittentadministration of an antisense nucleic acid or an antisense nucleicacid-GalNAc conjugate.

FIG. 16 shows the blood creatinine level in rats after intermittentadministration of an antisense nucleic acid or an antisense nucleicacid-GalNAc conjugate.

FIG. 17 shows the results of the comparison of the target geneexpression levels in mouse livers after single administration of twotypes of antisense nucleic acid-GalNAc conjugates using differentmain-chain linker structures for GalNAc connection.

DESCRIPTION OF EMBODIMENTS

The antisense nucleic acid of the present invention comprises anoligonucleotide and two or more linearly connected asialoglycoproteinreceptor-binding molecules attached to the oligonucleotide, wherein theoligonucleotide comprises a locked nucleoside analog having a bridgingstructure between the 4′ and 2′ positions, is complementary to a humanPCSK9 gene, and has inhibitory activity on the expression of the humanPCSK9 gene. Hereinafter, the antisense nucleic acid having theabove-mentioned characteristics may be referred to as an oligonucleotideconjugate of the present invention or an oligonucleotide conjugate.

Various studies have been performed on modification of nucleic acids byasialoglycoprotein (ASGP) receptor-binding molecules for liver-targetingdelivery of the nucleic acids. This approach is based on the findingthat, taking N-acetylgalactosamine (GalNAc) as an example, three GalNAcunits bind to an ASGP receptor on hepatic parenchymal cells whilemaintaining a specific conformation. Therefore, in order that thespecific conformation can be maintained, nucleic acids are usuallymodified by conjugation with tri-antennary GalNAc (three GalNAc unitseach attached to the same point). However, in the present invention,even nucleic acids modified by conjugation with linearly connected ASGPreceptor-binding molecules are shown to be sufficiently incorporatedinto hepatic parenchymal cells and exert favorable actions in vivo. Theprecise mechanism of this effect is unclear, but possible explanationsinclude the following. Due to the linear connection of the ASGPreceptor-binding molecules, the linker structure between the ASGPreceptor-binding molecules and the oligonucleotide is highly flexible;each of the connected ASGP receptor-binding molecules can flexibly fitin a spatially advantageous position of the ASGP receptor; and thepattern of metabolic transformation is advantageous for exerting theactivity of the oligonucleotide. This can give rise to improvedefficiency of uptake of the conjugates into the target cells, whichtogether with high biostability of the conjugates can contribute toenhanced inhibitory effect on the target gene expression, namely, humanPCSK9 gene expression. However, the putative mechanism described aboveshould not be construed as limiting the present invention.

The terms used herein will be defined as follows.

As used herein, the term “straight-chain alkyl group of 1 to 6 carbonatoms” usually refers to a straight-chain alkyl group of 1 to 6 carbonatoms, for example, a methyl group, an ethyl group, a n-propyl group, an-butyl group, a n-pentyl group, a n-hexyl group, or the like.

As used herein, the term “straight-chain alkoxy group of 1 to 6 carbonatoms” usually includes alkoxy groups having a straight-chain alkylgroup of 1 to 6 carbon atoms. For example, a methyloxy group, anethyloxy group, a n-propyloxy group, etc. are included.

As used herein, the term “straight-chain alkylthio group of 1 to 6carbon atoms” usually includes alkylthio groups having a straight-chainalkyl group of 1 to 6 carbon atoms. For example, a methylthio group, anethylthio group, a n-propylthio group, etc. are included.

As used herein, the term “straight-chain alkylamino group of 1 to 6carbon atoms” usually includes alkylamino groups having one or twostraight-chain alkyl groups of 1 to 6 carbon atoms. For example, amethylamino group, a dimethylamino group, an ethylamino group, amethylethylamino group, a diethylamino group, etc. are included.

As used herein, the term “optionally branched or cyclic alkyl group of 1to 7 carbon atoms” usually includes straight-chain alkyl groups of 1 to7 carbon atoms, branched-chain alkyl groups of 3 to 7 carbon atoms, andcyclic alkyl groups of 3 to 7 carbon atoms. These may be collectivelyreferred to simply as “a lower alkyl group”. Examples of thestraight-chain alkyl group of 1 to 7 carbon atoms include a methylgroup, an ethyl group, a n-propyl group, a n-butyl group, a n-pentylgroup, a n-hexyl group, and a n-heptyl group. Examples of thebranched-chain alkyl group of 3 to 7 carbon atoms include an isopropylgroup, an isobutyl group, a tert-butyl group, an isopentyl group, etc.Examples of the cyclic alkyl group of 3 to 7 carbon atoms include acyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc.

As used herein, the term “optionally branched or cyclic alkenyl group of2 to 7 carbon atoms” usually includes straight-chain alkenyl groups of 2to 7 carbon atoms, branched-chain alkenyl groups of 3 to 7 carbon atoms,and cyclic alkenyl groups of 3 to 7 carbon atoms. These may becollectively referred to simply as “a lower alkenyl group”. Examples ofthe straight-chain alkenyl group of 2 to 7 carbon atoms include anethenyl group, a 1-propenyl group, a 2-propenyl group, a 1-butenylgroup, a 2-butenyl group, a 1-pentenyl group, a 2-pentenyl group, a3-pentenyl group, a 4-pentenyl group, a 1-hexenyl group, etc. Examplesof the branched-chain alkenyl group of 3 to 7 carbon atoms include anisopropenyl group, a 1-methyl-1-propenyl group, a 1-methyl-2-propenylgroup, a 2-methyl-1-propenyl group, a 2-methyl-2-propenyl group, a1-methyl-2-butenyl group, etc. Examples of the cyclic alkenyl group of 3to 7 carbon atoms include a cyclobutenyl group, a cyclopentenyl group, acyclohexenyl group, etc.

As used herein, the term “aryl group of 3 to 12 carbon atoms which mayhave a heteroatom” usually includes aromatic hydrocarbons of 6 to 12carbon atoms composed of a hydrocarbon alone and heteroaromaticcompounds of 3 to 12 carbon atoms having a heteroatom (a nitrogen atom,an oxygen atom, a sulfur atom) in the ring structure. Examples of thearomatic hydrocarbon of 6 to 12 carbon atoms composed of a hydrocarbonalone include a phenyl group, a naphthyl group, an indenyl group, anazulenyl group, etc. Examples of the heteroaromatic compound of 3 to 12carbon atoms having a heteroatom in the ring structure include a pyridylgroup, a pyrrolyl group, a quinolyl group, an indolyl group, animidazolyl group, a furyl group, a thienyl group, etc.

As used herein, the term “alkyl group having an aryl moiety of 3 to 12carbon atoms which moiety may have a heteroatom” includes, for example,a benzyl group, a phenethyl group, a naphthylmethyl group, a3-phenylpropyl group, a 2-phenylpropyl group, a 4-phenylbutyl group, a2-phenylbutyl group, a pyridylmethyl group, an indolylmethyl group, afurylmethyl group, a thienylmethyl group, a pyrrolylmethyl group, a2-pyridylethyl group, a 1-pyridylethyl group, a 3-thienylpropyl group,etc. The total number of carbon atoms in such an alkyl group is notparticularly limited and is, for example, in the range of 4 to 18.

As used herein, the term “halogen atom” includes, for example, afluorine atom, a chlorine atom, a bromine atom, and an iodine atom.Preferred are a fluorine atom and a chlorine atom.

As used herein, the term “nucleoside” usually means a glycosylaminecomprising a nucleobase and a sugar. Examples of the nucleoside include,but are not limited to, a naturally-occurring nucleoside, an abasicnucleoside, a modified nucleoside, and a nucleoside having a pseudo-baseand/or a pseudo-sugar group.

As used herein, the term “nucleotide” usually means a glycosominecomprising a nucleobase and a sugar covalently bound to a phosphategroup. The nucleotide may be modified by a substituting group.

As used herein, the term “deoxyribonucleotide” usually means anucleotide having a hydrogen atom at the 2′ position of the sugar moietyof the nucleotide. The deoxyribonucleotide may be modified by asubstituting group.

As used herein, the term “deoxyribonucleic acid (DNA)” usually means anucleic acid composed of deoxyribonucleotides.

As used herein, the term “ribonucleotide” usually means a nucleotidehaving a hydroxyl group at the 2′ position of the sugar moiety of thenucleotide. The ribonucleotide may be modified by a substituting group.

As used herein, the term “ribonucleic acid (RNA)” usually means anucleic acid composed of ribonucleotides.

As used herein, the term “modified nucleoside” usually means anunnatural “nucleoside” in which a sugar is bound to a purine base or apyrimidine base; and a compound in which a sugar is bound to aheteroaromatic ring or aromatic hydrocarbon ring that is neither apurine base nor a pyrimidine base and is substitutable for a purine baseor a pyrimidine base. Preferred is a modified nucleoside havingmodification in the sugar moiety.

As used herein, the term “oligonucleotide” refers to an“oligonucleotide” having 2 to 50 identical or different “nucleosides”connected by one or more phosphodiester linkages or otherinternucleoside linkages. Unnatural derivatives of the “oligonucleotide”are also included. Preferable examples of such a derivative includeoligonucleotide derivatives in which at least one sugar moiety ismodified; oligonucleotide derivatives in which at least onephosphodiester moiety is converted to a phosphorothioate;oligonucleotide derivatives in which an oxygen atom of the phosphategroup of at least one phosphodiester linkage is substituted by a sulfuratom (phosphorothioate oligonucleotides); oligonucleotide derivatives inwhich the terminal phosphoric acid moiety is esterified; andoligonucleotide derivatives in which the amino group of at least onepurine base is amidated. The “oligonucleotide” includes asingle-stranded DNA or RNA and a double-stranded DNA or RNA unlessotherwise specified.

Preferred is a natural or unnatural single-stranded antisenseoligonucleotide. The “oligonucleotide” includes pharmaceuticallyacceptable salts thereof unless otherwise specified.

Next, the present invention will be described in more detail.

The oligonucleotide of the oligonucleotide conjugate of the presentinvention comprises a locked nucleoside analog having a bridgingstructure between the 4′ and 2′ positions. Due to the bridgingstructure, the oligonucleotide conjugate is resistant to degradationmediated by various nucleases and can stay in a living body for aprolonged time after administration.

An example of the bridging structure is a structure represented by—CH₂—O— or —(CH₂)₂—O—. Hereinafter, this bridging structure may bereferred to as a bridging structure of embodiment 1 (BNA).

Examples of the bridging structure of embodiment 1 include, but are notlimited to, α-L-methyleneoxy (4′-CH₂—O-2′, this may be referred to as“LNA”), β-D-methyleneoxy (4′-CH₂—O-2′), and ethyleneoxy(4′-(CH₂)₂—O-2′). The BNA nucleoside (monomer) or an oligonucleotidecomprising the BNA nucleosides can be synthesized by the methoddescribed in known literature, for example, WO 2011/052436.

Another example of the bridging structure is a structure represented by—CH₂—NR¹—O— or —(CH₂)₂—NR¹—O—. In the formula, R¹ represents a hydrogenatom;

an optionally branched or cyclic alkyl group of 1 to 7 carbon atoms;an optionally branched or cyclic alkenyl group of 2 to 7 carbon atoms;an aryl group of 3 to 12 carbon atoms which may have a heteroatom andmay have any one or more substituting groups selected from group αconsisting of a hydroxyl group, a straight-chain alkyl group of 1 to 6carbon atoms, a straight-chain alkoxy group of 1 to 6 carbon atoms, amercapto group, a straight-chain alkylthio group of 1 to 6 carbon atoms,an amino group, a straight-chain alkylamino group of 1 to 6 carbonatoms, and a halogen atom; or an alkyl group having an aryl moiety of 3to 12 carbon atoms which moiety may have a heteroatom and may have anyone or more substituting groups selected from the group α.

Hereinafter, this bridging structure may be referred to as a bridgingstructure of embodiment 2 (BNA^(NC)).

Examples of the bridging structure of embodiment 2 include, but are notlimited to, oxyamino (4′-CH₂—NH—O-2′) and N-methyloxyamino(4′-CH₂—NCH₃—O-2′). The BNA^(NC) nucleoside (monomer) or anoligonucleotide comprising the BNA^(NC) nucleosides can be synthesizedby the method described in known literature, for example, WO2011/052436.

Yet another example of the bridging structure is a structure representedby —CO—NR¹—, —CH₂—CO—NR¹—, —(CH₂)₂—CO—NR¹—, —CO—NR¹—X—, or—CH₂—CO—NR¹—X—. In the formula, R¹ represents a hydrogen atom;

an optionally branched or cyclic alkyl group of 1 to 7 carbon atoms;an optionally branched or cyclic alkenyl group of 2 to 7 carbon atoms;an aryl group of 3 to 12 carbon atoms which may have a heteroatom andmay have any one or more substituting groups selected from group αconsisting of a hydroxyl group, a straight-chain alkyl group of 1 to 6carbon atoms, a straight-chain alkoxy group of 1 to 6 carbon atoms, amercapto group, a straight-chain alkylthio group of 1 to 6 carbon atoms,an amino group, a straight-chain alkylamino group of 1 to 6 carbonatoms, and a halogen atom; or an alkyl group having an aryl moiety of 3to 12 carbon atoms which moiety may have a heteroatom and may have anyone or more substituting groups selected from the group α, andX represents an oxygen atom, a sulfur atom, an amino group, or amethylene group.

Hereinafter, this bridging structure may be referred to as a bridgingstructure of embodiment 3 (AmNA).

Examples of the bridging structure of embodiment 3 include, but are notlimited to, non-substituted amide (4′-CO—NH-2′), N-methylamide(4′-CO—NCH₃-2′), acetamide (4′-CH₂—CO—NH-2′), N-methylacetamide(4′-CH₂—CO—NCH₃-2′), N-oxyacetamide (4′-CH₂—CO—NH—O-2′), andN-methyl-N-oxyacetamide (4′-CH₂—CO—NCH₃—O-2′). The AmNA nucleoside(monomer) or an oligonucleotide comprising the AmNA nucleosides can besynthesized by the method described in known literature, for example, WO2012/029870.

Yet still another example of the bridging structure is a structurerepresented by —CH₂—NR¹— or —(CH₂)₂—NR¹—. In the formula, R¹ representsa hydrogen atom;

an optionally branched or cyclic alkyl group of 1 to 7 carbon atoms;an optionally branched or cyclic alkenyl group of 2 to 7 carbon atoms;an aryl group of 3 to 12 carbon atoms which may have a heteroatom andmay have any one or more substituting groups selected from group αconsisting of a hydroxyl group, a straight-chain alkyl group of 1 to 6carbon atoms, a straight-chain alkoxy group of 1 to 6 carbon atoms, amercapto group, a straight-chain alkylthio group of 1 to 6 carbon atoms,an amino group, a straight-chain alkylamino group of 1 to 6 carbonatoms, and a halogen atom; or an alkyl group having an aryl moiety of 3to 12 carbon atoms which moiety may have a heteroatom and may have anyone or more substituting groups selected from the group α.

Hereinafter, this bridging structure may be referred to as a bridgingstructure of embodiment 4.

Examples of the bridging structure of embodiment 4 include, but are notlimited to, amino (4′-CH₂—NH-2′) and N-methylamino (4′-CH₂—NCH₃-2′). Anucleoside (monomer) having the bridging structure of embodiment 4 or anoligonucleotide comprising the nucleosides can be synthesized by themethod described in known literature, for example, Kumar R. et al.,Bioorg. & Med. Chem. Lett., 1998, 8, 2219-2222; or Singh S. K. et al.,J. Org. Chem., 1998, 63, 10035-39.

The locked nucleoside analog can be prepared by introducing any of thesebridging structures into a nucleoside as the monomeric unit of anoligonucleotide. In the case where the oligonucleotide contains two ormore locked nucleoside analogs, the bridging structures may be all thesame or different. There is no particular limitation. That is, two ormore types of bridging structures of the same embodiment may be used incombination, and two or more types of bridging structures of differentembodiments may be used in combination. There is no particularlimitation.

The percentage of the number of locked nucleoside analogs in theoligonucleotide used in the present invention is not particularlylimited. For example, the lower limit is 5%, 7%, 10%, 15%, 20%, or 25%of the total number. In addition, the upper limit is also notparticularly limited. For example, the oligonucleotide used in thepresent invention may be exclusively composed of locked nucleosideanalogs, in other words, the percentage of the number of lockednucleoside analogs in the oligonucleotide may be 100%. Also, the upperlimit may be 90%, 80%, 70%, or 60% of the total number.

The location of the locked nucleoside analog in the oligonucleotide isnot particularly limited. For example, the locked nucleoside analog maybe located at the 5′- or 3′-end or both ends of the oligonucleotide.Alternatively, more than one locked nucleotide analog may be locateddiscontinuously or contiguously in the oligonucleotide. For example, twocontiguous nucleosides from the 5′-end and the 2nd and 3rd nucleosidesfrom the 3′-end may be locked nucleoside analogs.

The oligonucleotide used in the present invention is complementary to ahuman PCSK9 gene and capable of binding to the gene. The term “capableof binding” means that two or more different single-strandedoligonucleotides or nucleic acids can form a two or more-strandednucleic acid due to the complementarity of base pairs between the two ormore strands. Preferably, the term “capable of binding” means that twodifferent single-stranded oligonucleotides or nucleic acids can form adouble-stranded nucleic acid. The melting temperature (Tm) of the two ormore-stranded nucleic acid is not particularly limited. For example,when two different single-stranded oligonucleotides or nucleic acidsform a double-stranded nucleic acid, both the nucleotide sequences donot have to be completely complementary in the double-stranded region.

The oligonucleotide used in the present invention has inhibitoryactivity on human PCSK9 gene expression. More specifically, for example,the oligonucleotide used in the present invention forms a stabledouble-stranded structure with human PCSK9 mRNA and degrades PCSK9 mRNAand/or inhibits the biosynthesis of PCSK9 protein. In the presentinvention, such inhibitory activity can be measured and evaluated usinga technique called the “Ca²⁺ enrichment of medium (CEM) method”.

The procedure for evaluation of the inhibitory activity using the CEMmethod is as follows. A human hepatoma cell line, Huh-7, is cultured ina usual medium for 24 hours. After that, the medium is replaced with CEM(CaCl₂)-containing medium) containing a test oligonucleotide at 200 nM.After 24 hours, total RNA is extracted, and the expression level ofPCSK9 mRNA is quantified on a real-time PCR system. A lower expressionlevel of PCSK9 mRNA indicates that the test oligonucleotide has higherinhibitory activity on human PCSK9 gene expression.

The human PCSK9 gene comprises the nucleotide sequence of SEQ ID NO: 1(GenBank accession number: NM 174936; coding region, 2079 bases) andencodes the amino acid sequence of SEQ ID NO: 2. The PCSK9 gene plays arole in LDL receptor degradation. As used herein, the human PCSK9 geneincludes not only a gene consisting of the nucleotide sequence of SEQ IDNO: 1, but also a variant thereof which may occur in a human body, forexample, a variant gene consisting of a nucleotide sequence identical tothe nucleotide sequence of SEQ ID NO: 1 except for one to several basedeletions, substitutions, and/or additions due to polymorphism orspontaneous mutation. Moreover, the human PCSK9 gene includes a variantconsisting of a nucleotide sequence which has, for example, 80% or more,85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% ormore, 99.5% or more, or 99.9% or more identity to the nucleotidesequence of SEQ ID NO: 1. The identity of the nucleotide sequence can bedetermined using a known algorithm such as BLAST or FASTA.

The region of the human PCSK9 gene to which the oligonucleotide used inthe present invention is capable of binding is preferably represented bya nucleotide sequence comprising any of the following: the nucleotidesequence of SEQ ID NO: 3; the nucleotide sequence of SEQ ID NO: 4; thenucleotide sequence of SEQ ID NO: 5; the nucleotide sequence of SEQ IDNO: 6; the nucleotide sequence of SEQ ID NO: 7; the nucleotide sequenceof SEQ ID NO: 8; the nucleotide sequence of SEQ ID NO: 9; the nucleotidesequence of SEQ ID NO: 10; the nucleotide sequence of SEQ ID NO: 11; thenucleotide sequence of SEQ ID NO: 12; the nucleotide sequence of SEQ IDNO: 13; the nucleotide sequence of SEQ ID NO: 14; and complementarynucleotide sequences thereof. More preferred is a DNA or RNA consistingof any of these nucleotide sequences.

The oligonucleotide used in the present invention can be synthesized bythe usual method. For example, the oligonucleotide can readily besynthesized with a commercial DNA synthesizer (e.g., manufactured byThermo Fisher Scientific, etc.). The synthesis method may be aphosphoramidite-based solid phase synthesis method, ahydrogenphosphonate-based solid phase synthesis method, or the like.

The length of the nucleotide sequence of the oligonucleotide used in thepresent invention is not particularly limited. For example, theoligonucleotide used in the present invention preferably has a 10- to25-base nucleotide sequence and more preferably has a 13- to 20-basenucleotide sequence. The internucleoside linkage is, for example, aphosphodiester linkage or another type of internucleoside linkage (e.g.,a phosphorothioate linkage). Preferred is a phosphorothioate linkagebecause it is advantageous for inhibition of PCSK9 expression.

In the present invention, the oligonucleotide conjugate is characterizedin that two or more linearly connected ASGP receptor-binding moleculesare attached to the 5′- or 3′-end or both ends of the oligonucleotide.The term “attached to both ends” herein means that a set of two or morelinearly connected ASGP receptor-binding molecules is attached to eachend. The above characteristic enables targeting delivery of theoligonucleotide conjugate of the present invention to hepaticparenchymal cells.

The ASGP receptor-binding molecule refers to a molecule capable ofbinding to an ASGP receptor and is, for example, an asialoglycoprotein.Specific examples of the asialoglycoprotein include lactose, galactose,N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine,N-propionylgalactosamine, N-n-butanoylgalactosamine,N-iso-butanoylgalactosamine, and derivatives thereof. The derivative ofthe asialoglycoprotein is not particularly limited as long as it iscapable of binding to an ASGP receptor. Examples include derivatives ofasialoglycoproteins obtained by functional group conversion etc.; andasialoglycoproteins substituted with saccharides, amino acids, vitamins,or fatty acids. Also included are low-molecular-weight compounds withouta sugar backbone; monoclonal antibodies against an ASGP receptor(including monoclonal antibody fragments and antibody-like moleculessuch as ankyrin); and nucleic acid aptamers. The ASGP receptor-bindingmolecules attached to the oligonucleotide may be a single type ofmolecule or a combination of two or more types of molecules selectedfrom the foregoing examples.

As long as two or more ASGP receptor-binding molecules are attached tothe 5′- or 3′-end or both ends of the oligonucleotide, there is noparticular limitation on the number of the ASGP receptor-bindingmolecules attached thereto. Preferred is 3 or more. In addition, thenumber of the ASGP receptor-binding molecules is preferably 10 or less,more preferably 7 or less, and still more preferably 5 or less. In thecase where the ASGP receptor-binding molecules are attached to both endsof the oligonucleotide, the total number of the ASGP receptor-bindingmolecules attached thereto is not particularly limited and is, forexample, 4 to 20. The linkage between the ASGP receptor-bindingmolecules is, for example, a phosphodiester linkage or aphosphorothioate linkage, but preferred is a phosphodiester linkagebecause the conjugate of the present invention properly undergoesintracellular metabolism that enables the oligonucleotide to efficientlyact on the target mRNA.

The ASGP receptor-binding molecules may be attached to theoligonucleotide via a linker. Specifically, for example, in the casewhere two or more ASGP receptor-binding molecules are attached to theoligonucleotide, two or more main-chain linkers are connected andattached to the oligonucleotide; and each ASGP receptor-binding moleculeis attached to a side-chain linker branched from each main-chain linker.The main-chain linker is not particularly limited and is, for example, astraight-chain or branched-chain, saturated or unsaturated carbon chainspacer. In the case where the side-chain linker contains a heteroatom asdescribed later, the heteroatom together with some carbon atoms in thecarbon chain of the main-chain linker may form a heterocycle. The lengthof the carbon chain is not particularly limited, but in terms of theflexibility of the ASGP receptor-binding molecules for binding to anASGP receptor, the lower limit of the number of carbon atoms ispreferably 2, and the upper limit is, for example, 18, 16, 12, 10, 8, 6,5, or 4. Specific examples of the carbon chain include, an ethylenechain, a propylene chain, a butylene chain, an isopropylene chain, apentylene chain, a hexylene chain, a heptylene chain, an octylene chain,a nonylene chain, a decylene chain, a dodecylene chain, a tetradecylenechain, a hexadecylene chain, an octadecylene chain, etc. The two or moremain-chain linkers in the conjugate may be the same or different. Theside-chain linker is also not particularly limited and is, for example,a straight-chain or branched-chain, saturated or unsaturated carbonchain spacer (optionally containing a heteroatom or a heterocycle). Thelength of the carbon chain is not particularly limited, and the numberof carbon atoms is, for example, about 5 to 50. The main-chain linkerand the side-chain linker may be collectively referred to simply as a“linker”. The linker in the present invention preferably has a highlyflexible structure so that proper metabolism of the conjugate of thepresent invention can be facilitated within cells. In addition, thelinker preferably has such a structure that each of the connected ASGPreceptor-binding molecules flexibly fits in a spatially advantageousposition of the ASGP receptor. The linker having such a structure allowsconnection of ASGP receptor-binding molecules while maintaining theirindividual flexibility. For example, a main-chain linker that is astraight-chain saturated carbon chain is more flexible than a main-chainlinker that has a cyclic structure. The linkage between theoligonucleotide and the ASGP receptor-binding molecules, that is, thelinkage between the oligonucleotide and the linker is, for example, aphosphodiester linkage or a phosphorothioate linkage, but preferred is aphosphodiester linkage because the conjugate of the present inventionproperly undergoes intracellular metabolism that enables theoligonucleotide to efficiently act on the target mRNA. Hereinafter,described are embodiments where a preferable linker is used forconnection of the ASGP receptor-binding molecules.

(A) an embodiment in which each ASGP receptor-binding molecule bound toa butylene-based main-chain linker via a side-chain linker which islinked to the main-chain linker through a pyrrolidine ring (hereafterreferred to as structure (A)); and(B) an embodiment in which each ASGP receptor-binding molecule bound toan ethylene-based main-chain linker via a side-chain linker (hereafterreferred to as structure (B)).

The present invention includes embodiments where a linker structurecomposed of a combination of structures (A) and (B) is used.

The attachment of two or more connected ASGP receptor-binding moleculesto the oligonucleotide can be performed according to the methoddescribed in known literature, for example, Yamamoto T et al., BioorgMed Chem., 2016, 24, 26-32.

In addition to terminal modification of the oligonucleotide by the ASGPreceptor-binding molecules, the oligonucleotide conjugate of the presentinvention may have a known chemical modification of nucleotides asmonomeric units. Specifically, for example, additional ASGPreceptor-binding molecules and/or other functional molecules may beintroduced into the 2′-nitrogen atom of AmNA, the 2′-nitrogen atom ofBNA^(NC), the position 5′ of uracil, etc., via a known linker as needed.Such modifications can alter the activity of the oligonucleotide, forexample, enhance the affinity for the target nucleic acid, increasenuclease resistance, reduce off-target toxicity, and/or alter thepharmacokinetics or tissue distribution of the oligonucleotide. Theposition and number of such modifications are not particularly limitedand may be determined as appropriate for the purposes.

In addition to 5′- or 3′-terminal modification of the oligonucleotide bythe ASGP receptor-binding molecules, the oligonucleotide conjugate ofthe present invention may have at least one additional componentattached thereto. The at least one additional component is, for example,selected from the group consisting of sugars such as mannose,antibodies, aptamers, intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic-acid moieties, folic acid, lipids,phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluorescein, rhodamine, coumarin, and pigments.The attachment of the at least one additional component can be achievedaccording to a known method. The at least one additional component maybe attached to the ASGP receptor-binding molecules attached to the 5′-or 3′-end of the oligonucleotide.

The oligonucleotide conjugate of the present invention can be in theform of a pharmaceutically acceptable salt, ester or ester salt, or inanother derivative form which is capable of providing, either directlyor indirectly, a biologically active metabolite or residue onceadministered to animals including humans.

The pharmaceutically acceptable salt refers to a salt of theoligonucleotide conjugate of the present invention acceptable forphysiological and pharmaceutical use, namely, a salt which retains thedesired biological activity of a parent compound without unwantedtoxicological effects. Preferable examples of the pharmaceuticallyacceptable salt of an oligonucleotide and use thereof are well known tothe skilled person.

Specific preferable examples of the pharmaceutically acceptable salt ofan oligonucleotide include, but are not limited to, (a) salts formedwith cations such as sodium, potassium, ammonium, magnesium, calcium,and polyamines such as spermine and spermidine; (b) acid addition saltsformed with inorganic acids, for example, hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, and nitric acid; (c) salts formedwith organic acids such as acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, and polygalacturonic acid; and (d) salts formed with anions ofelements such as chlorine, bromine, and iodine.

The features of the oligonucleotide conjugate of the present inventionare as described above. In an exemplary embodiment, the oligonucleotidehas a 14-base nucleotide sequence; two contiguous nucleosides from the5′-end of the oligonucleotide and the 2nd and 3rd nucleosides from the3′-end of the oligonucleotide are locked nucleoside analogs each havinga bridging structure of embodiment 1; internucleoside linkages arephosphorothioate linkages; and three N-acetylgalactosamine molecules arelinearly connected and attached to the 5′-end of the oligonucleotide. Inthis embodiment, the linkages between the three N-acetylgalactosaminemolecules and the linkage between the oligonucleotide and theN-acetylgalactosamine molecules are phosphodiester linkages, and thethree N-acetylgalactosamine molecules may be connected by phosphodiesterlinkages via linkers.

The oligonucleotide conjugate of the present invention, which has theabove-described bridging structure and modification by ASGPreceptor-binding molecules, can be stably incorporated into target cellsand play a functional role therein. For example, the oligonucleotideconjugate can form a stable double-stranded structure with mRNA of apathogenic protein and inhibit biosynthesis of the protein (theantisense method). Also, the oligonucleotide conjugate can form athree-stranded structure with genomic double-stranded DNA and inhibitmRNA transcription. For these reasons, the oligonucleotide conjugate ofthe present invention is potentially useful as a pharmaceutical product(antisense nucleic acid) that blocks the action of the PCSK9 gene fordisease therapy. More specifically, for example, the oligonucleotideconjugate of the present invention is a promising antisense nucleic acidthat binds to human PCSK9 mRNA and inhibits human PCSK9 gene expression.The inhibition of human PCSK9 gene expression leads to increase in LDLreceptor protein expression level and subsequent enhancement in LDLcellular uptake and metabolism, resulting in reduction in blood LDLlevel. Thus, the oligonucleotide conjugate of the present inventionserves as a dyslipidemia therapeutic agent, for example. Hereinafter, apharmaceutical product comprising the oligonucleotide conjugate of thepresent invention as an active ingredient is referred to as apharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention can specificallyinhibit PCSK9, and therefore, can be used as a preventive or therapeuticagent for a disease associated with a high LDL cholesterol level.Examples of the disease associated with a high LDL cholesterol levelinclude dyslipidemia such as hypercholesterolemia including familialhypercholesterolemia. Also included are high-risk diseases in more needof LDL cholesterol reduction, such as a history of coronary arterydisease, diabetes, chronic nephropathy, noncardiogenic cerebralinfarction, and peripheral arterial disease.

The pharmaceutical composition of the present invention can be preparedas a parenteral preparation or a liposome preparation by blending theoligonucleotide conjugate of the present invention with an adjuvantusually used in the technical field of pharmaceutical preparations, suchas a filler, a binder, a preservative, an oxidation stabilizer, adisintegrant, a lubricant, and a taste masking agent. Examples of theparenteral preparation include a transpulmonary preparation (e.g., apreparation for use with a nebulizer etc.), a transnasal preparation, atransdermal preparation (e.g., an ointment, a cream), an injectablepreparation, etc. The injectable preparation can be administered locallyor systemically by, for example, intravenous injection such as infusion,intramuscular injection, intraperitoneal injection, subcutaneousinjection, or the like. The injectable preparation can be produced as asolution or a lyophilized preparation for reconstitution before use byblending the oligonucleotide conjugate of the present invention with apharmaceutical carrier usually used in the technical field.

The dose of the pharmaceutical composition of the present invention mayvary with the age, the sex, the symptom, the route and frequency ofadministration, and the dosage form. The mode of administration can beselected as appropriate for patient's age and symptom. The effectivedose includes, for example, 0.01 μg to 1000 mg, 0.1 μg to 100 μg, or thelike of the oligonucleotide conjugate of the present invention peradministration to a human weighing 50 kg.

The individual suitable for the application of the pharmaceuticalcomposition of the present invention is preferably a human in need oftherapy for a disease associated with a high LDL cholesterol level, butis not limited thereto. For example, a pet animal in need of therapy fora disease associated with a high LDL cholesterol level is also suitable.The disease associated with a high LDL cholesterol level is as describedabove.

The present invention also provides the following embodiments. Thespecifications of the oligonucleotide conjugate of the presentinvention, the preparation method thereof, and the like are as describedabove in the section for describing the oligonucleotide conjugate of thepresent invention.

(I) A method for treating a disease associated with a high LDLcholesterol level, the method comprising a step of administering theoligonucleotide conjugate of the present invention.(II) Use of the oligonucleotide conjugate of the present invention fortreatment of a disease associated with a high LDL cholesterol level.(III) Use of the oligonucleotide conjugate of the present invention forproduction of a therapeutic agent for a disease associated with a highLDL cholesterol level.

EXAMPLES

Hereinafter, the present invention will be described in detail withreference to examples which are illustrative only and should not beconstrued as limiting the invention.

Test Example 1

A human hepatoma cell line, Huh-7, was seeded on plates and cultured inDMEM (10% FBS, 1% penicillin, 1% streptomycin) for 24 hours. After that,the medium was replaced with CEM (DMEM containing 10% FBS, 1%penicillin, 1% streptomycin, and 9 mM CaCl₂)) containing a testantisense oligonucleotide at 200 nM. After 24 hours, total RNA wasextracted and then subjected to cDNA synthesis. Gene amplification wasperformed on a StepOnePlus (registered trademark) real-time PCR system(ABI) using the probes shown below to quantify the expression levels ofPCSK9 mRNA and GAPDH mRNA. The relative expression level of PCSK9 mRNAwas then determined. For this test, a variety of antisenseoligonucleotides were prepared by introducing an “LNA (4′-CH₂—O-2′)” or“AmNA (4′-CO—NCH₃-2′)” bridging structure into the nucleotide sequencesof selected regions of the human PCSK9 gene according to a known method.The results are shown in FIG. 1.

PCSK9: Hs00545399_m1 (Thermo Fisher Scientific, Product No. 4331182)GAPDH: Hs02786624_g1 (Thermo Fisher Scientific, Product No. 4331182)

As shown in FIG. 1, the relative expression level of PCSK9 mRNA wasbelow 0.05 in some antisense oligonucleotides. The results demonstratethat the CEM method can be used to efficiently screen for antisenseoligonucleotides having inhibitory effect on PCSK9 gene expression. Thesequences of the antisense oligonucleotides shown to have inhibitoryeffect are specifically listed below. All the listed antisenseoligonucleotides having a bridging structure, whether LNA or AmNA,showed lower relative expression levels of PCSK9 mRNA, indicating thatthe antisense oligonucleotides having the same sequence have similaractivities regardless of the type of the bridging structure used intheir sequences.

TABLE 1 Name of Nucleotide oligo- sequence of Target region nucleotideoligonucleotide of PCSK9 gene HsPCSK9-61 5′-GGacccaggagCAg-3′  423-436(SEQ ID NO: 3) HsPCSK9-311 5′-GAggtatccccGGc-3′  673-686 (SEQ ID NO: 4)HsPCSK9-591 5′-CCatgaccctgCCc-3′  953-966 (SEQ ID NO: 5) HsPCSK9-6615′-CTgtcacacttGCt-3′ 1023-1036 (SEQ ID NO: 6) HsPCSK9-8715′-CGgctgtacccACc-3′ 1233-1246 (SEQ ID NO: 7) HsPCSK9-10915′-GAtgtcctcccCTg-3′ 1453-1466 (SEQ ID NO: 8) HsPCSK9-11315′-GTgacacaaagCAg-3′ 1493-1506 (SEQ ID NO: 9) HsPCSK9-11715′-ATgccagccacGTg-3′ 1533-1546 (SEQ ID NO: 10) HsPCSK9-13515′-AGctgccaaccTGc-3′ 1713-1726 (SEQ ID NO: 11) HsPCSK9-13815′-GAgtgtgctgaCCa-3′ 1743-1756 (SEQ ID NO: 12) HsPCSK9-17715′-CTggcctccctGTg-3′ 2133-2146 (SEQ ID NO: 13) HsPCSK9-18115′-GCattccagacCTg-3′ 2173-2186 (SEQ ID NO: 14) *All the internucleotidelinkages are phosphorothioate linkages. The upper-case letter indicatesfor DNA having LNA or AmNA, and the lower-case letter indicates DNA.

Test Example 2

A human hepatoma cell line, Huh-7, was seeded on plates and cultured inDMEM (10% FBS, 1% penicillin, 1% streptomycin) for 24 hours. After that,the medium was replaced with CEM (DMEM containing 10% FBS, 1%penicillin, 1% streptomycin, and 9 mM CaCl₂)) containing an antisenseoligonucleotide at a final concentration of 8 to 200 nM. In this test,antisense oligonucleotides selected based on the results of thescreening of Test Example 1 (antisense oligonucleotides having an AmNAbridging structure) were used. After 24 hours, total RNA was extractedand then subjected to cDNA synthesis. Gene amplification was performedon a StepOnePlus (registered trademark) real-time PCR system (ABI) usingSYBR Green (Fast SYBR (registered trademark) Green Master Mix) and thesame probes as used in Test Example 1 to determine the relativeexpression level of PCSK9 mRNA. The results are shown in FIG. 2.

As shown in FIG. 2, the concentration dependency was confirmed in allthe sequences. In particular, HsPCSK9-1811 (SEQ ID NO: 14) had thehighest activity. This antisense oligonucleotide was completelycomplementary to the rat and cynomolgus monkey PCSK9 genes, which wereused in the efficacy and safety tests described later. For thesereasons, this antisense oligonucleotide was selected for subsequenttests.

Test Example 3

HsPCSK9-1811 (SEQ ID NO: 14) having an LNA or AmNA bridging structurewas evaluated for efficacy in hyperlipidemic cynomolgus monkeys.

The specific procedure was as follows. Prior to the test, the LDLcholesterol levels of cynomolgus monkeys (purpose-bred, anti-B virusantibody negative, 2 to 4 years old, male) were measured to pre-selectanimals with high LDL cholesterol levels. From among the pre-selectedanimals, those with sustained high LDL cholesterol levels at 6 daysbefore administration were selected and used. Each antisense nucleicacid was subcutaneously administered to the selected cynomolgus monkeyson an intermittent schedule at increasing doses, namely, at 1 mg/kg atthe start of the test (day 0), at 3 mg/kg on day 7, and at 10 mg/kg onday 14. After that, in the case where significant reduction in blood LDLcholesterol level was observed, the same antisense nucleic acid wasadditionally administered at decreasing doses, namely, at 3 mg/kg on day42 and at 1 mg/kg on day 61. The blood LDL cholesterol level wasmeasured with an automated biochemical analyzer (JCA-BM6070, JEOL Ltd.)every 2 or 3 days from the start of the test until day 100. The bloodLDL cholesterol level was presented as a relative value compared to thatat the start of the test and evaluated. In addition, the blood PCSK9level after 10 mg/kg administration was measured using CircuLex(registered trademark) Human PCSK9 ELISA Kit (CycLex). The blood PCSK9level was presented as a relative value compared to that at the timepoint of 10 mg/kg administration and evaluated. The results are shown inFIG. 3 and FIG. 4.

As shown in FIG. 3, the blood LDL cholesterol level was reduced afterantisense administration regardless of the type of the bridgingstructure. In the case of the administration of the antisense nucleicacid having an LNA bridging structure, a continuous remarkable reduction(approximately 60% reduction) in blood LDL cholesterol level wasobserved for 28 days after 10 mg/kg administration. These results wereconsistent with the results in FIG. 4 showing reduction in blood PCSK9level, indicating that the antisense nucleic acid exerted its activity.In addition, the blood LDL cholesterol level was reversed byintermittent administration at decreasing doses. The above resultsdemonstrate that the antisense nucleic acid exerted its activity inhyperlipidemic cynomolgus monkeys although the potency varied with thetype of the bridging structure.

Test Example 4

HsPCSK9-1811 (SEQ ID NO: 14) having an LNA bridging structure wassubjected to a safety test on cynomolgus monkeys.

The specific procedure was as follows. The antisense nucleic acid wassubcutaneously administered to cynomolgus monkeys (purpose-bred, anti-Bvirus antibody negative, 3 to 4 years old, male) at a dose of 10 mg/kgor 30 mg/kg on an intermittent schedule, namely, once a week for 2 weeks(2 times in total) (10 mg/kg administration group: No. 1 and No. 2, 30mg/kg administration group: No. 3 and No. 4, n=2 per group). Afteradministration, the following observation and analysis were performed:general condition, body weight, feed consumption, water intake, urineanalysis, hematological analysis, blood biochemical analysis, necropsy,organ weight, and histopathological analysis. For the urine analysis, anautomated biochemical analyzer (JCA-BM6070, JEOL Ltd.) was used, and forthe blood biochemical analysis, an automated biochemical analyzer(JCA-BM6070, JEOL Ltd.) was used. The representative results are shownin FIGS. 5 to 8.

In both the 10 mg/kg and 30 mg/kg administration groups, renalhypertrophy accompanied by kidney weight gain were observed (FIG. 5),and elevated urinary protein levels were also detected by the urineanalysis (FIG. 6). In the blood biochemical analysis, elevated bloodurea nitrogen levels, which were indicative of renal disorder, wereobserved in both groups (FIG. 7), and an elevated blood creatinine levelwas observed in 30 mg/kg administration group (FIG. 8).

The above results indicate that the estimatedno-observed-adverse-effect-level (NOAEL) of HsPCSK9-1811-LNA(14) is lessthan 10 mg/kg. In Test Example 3 as well as the phase I trial of SPC5001on healthy volunteers, administration of an oligonucleotide alone at anefficacious dose caused adverse effects, in particular, serious renaldamage. This finding indicates the need for alteration ofoligonucleotides in terms of pharmacokinetics to achieve safer therapy.

Test Example 5

HsPCSK9-1811 (SEQ ID NO: 14) having an LNA bridging structure wassubjected to a preliminary toxicity test on rats.

More specifically, the antisense nucleic acid was subcutaneouslyadministered to rats (Crl:CD (SD), 6 weeks old, Charles RiverLaboratories Japan, Inc., five males and five females per group) at adose of 10 mg/kg or 30 mg/kg on an intermittent schedule, namely, once aweek for 2 weeks (2 times in total) (n=5). For the control group,physiological saline was subcutaneously administered to rats on the sameintermittent schedule as for the antisense-nucleic-acid administrationgroups. After administration, the following observation and analysiswere performed in the same manner as in Test Example 4: generalcondition, body weight, feed consumption, hematological analysis, bloodbiochemical analysis, necropsy, organ weight, and histopathologicalanalysis. The representative results are shown in FIG. 9 and FIG. 10.

Similarly to the results of Test Example 4, renal hypertrophyaccompanied by kidney weight gain was observed in both the 10 mg/kg and30 mg/kg administration groups; and elevated blood urea nitrogen levels(FIG. 9) and elevated blood creatinine levels (FIG. 10) were detected bythe blood biochemical analysis.

The above results indicate that the estimatedno-observed-adverse-effect-level (NOAEL) of HsPCSK9-1811-LNA(14) is lessthan 10 mg/kg. Similarly to the results of Test Example 4,administration of an oligonucleotide alone at an efficacious dose causedadverse effects, in particular, serious renal damage. This findingindicates the need for alteration of oligonucleotides in terms ofpharmacokinetics to achieve safer therapy.

Test Example 6

HsPCSK9-1811 (SEQ ID NO: 14) having an LNA bridging structure, whichcaused renal damage in Test Example 4 and Test Example 5, was modifiedby contiguously introducing three GalNAc monomeric units (amidatedGalNAc units) by the phosphoramidite method on an automatedoligonucleotide synthesizer (OligoPilot 10, GE Healthcare). Thus, anantisense nucleic acid-GalNAc conjugate (HsPCSK9-1811-LNA(14)-GN(3)) wasobtained. The schematic view of the 14-mer antisense nucleic acid-GalNAcconjugate is shown in FIG. 11. HsPCSK9-1811-LNA(14)-GN(3) was subjectedto an efficacy test on hyperlipidemic cynomolgus monkeys. For theattachment of the GalNAc units, the linkers of the above-describedstructure (A) were used and illustrated below.

The specific procedure was as follows. The antisense nucleic acid-GalNAcconjugate was subcutaneously administered to hyperlipidemic cynomolgusmonkeys (purpose-bred, anti-B virus antibody negative, 3 to 4 years old,male), which were selected in advance as described in Test Example 3, ata single dose of 0.3 mg/kg or 1 mg/kg (n=1). The blood LDL cholesterollevel was measured with an automated biochemical analyzer (JCA-BM6070,JEOL Ltd.) every 2 or 3 days from the start of the test until day 53.The blood LDL cholesterol level was presented as a relative valuecompared to that at the start of the test and evaluated. The results areshown in FIG. 12.

A remarkable reduction in blood LDL cholesterol level was observed atboth doses in hyperlipidemic cynomolgus monkeys. The results indicatethat the efficacious dose is about one-thirtieth to one-tenth of that ofHsPCSK9-1811-LNA(14).

Test Example 7

HsPCSK9-1811 (SEQ ID NO: 14) having an LNA bridging structure(HsPCSK9-1811-LNA(14)) or HsPCSK9-1811 (SEQ ID NO: 14) having an LNAbridging structure and a GalNAc modification(HsPCSK9-1811-LNA(14)-GN(3)) was subcutaneously administered to male6-week-old Crl:CD (SD) rats (5 animals per group) on an intermittentschedule for 2 weeks to examine whether there would be difference intoxicity between these oligonucleotides. The efficacious dose ofHsPCSK9-1811-LNA(14)-GN(3) was set at 0.3 mg/kg by reference to theresults of Test Example 6. The high dose was set at 3 mg/kg, which was10 times the efficacious dose, and the medium dose was set at 1 mg/kg.The dose of HsPCSK9-1811-LNA(14) was set at 30 mg/kg, at which renaldamage had been observed in Test Examples 4 and 5. In each case, theoligonucleotide was administered once a week for 2 weeks (2 times intotal). For the control group, physiological saline was subcutaneouslyadministered to rats on the same schedule as for the test substanceadministration groups. For blood biochemical analysis, an automatedbiochemical analyzer (JCA-BM6070, JEOL Ltd.) was used. The urinarykidney injury molecule (Kim-1) level was measured with Bio-Plex 200(Bio-Rad). The representative results are shown in FIGS. 13 to 16.

In the HsPCSK9-1811-LNA(14) administration group, renal hypertrophyaccompanied by kidney weight gain (FIG. 13) and an elevated bloodcreatinine level (FIG. 16) were observed and consistent with the resultsof Test Examples 4 and 5. The urine analysis showed an increase inurinary output (FIG. 14) and an elevated level of urinary kidney injurymolecule 1 (Kim-1), an early marker of acute renal damage (FIG. 15). Incontrast, the HsPCSK9-1811-LNA(14)-GN(3) administration groups did notshow the above change or elevated levels. The histopathological analysisshowed that the HsPCSK9-1811-LNA(14) administration group had changes inthe kidney, such as degeneration, necrosis, and regeneration in thetubular epithelium, tubular enlargement, hyaline cast formation, andmononuclear cell infiltration into the tubulointerstitium. In contrast,none of the above changes were observed at any dose ofHsPCSK9-1811-LNA(14)-GN(3).

Test Example 8

In order to examine whether the activity of an antisense nucleic acid inthe liver would be affected by the structure of a main-chain linker usedfor connection of GalNAc units, an antisense nucleic acid-GalNAcconjugate having linkers of the above-described structure (A) and anantisense nucleic acid-GalNAc conjugate having linkers of theabove-described structure (B) were prepared (referred to as conjugateA-I and conjugate B-I, respectively). Saline (physiological saline) oreach antisense nucleic acid-GalNAc conjugate was subcutaneouslyadministered to male 8-week-old wild-type mice (Japan SLC, Inc) at asingle dose of 17.5 nmol/kg. Three days after administration, liverswere excised, and the target gene expression levels were quantified on areal-time PCR system (ABI). The structure for the connection of GalNAcunits is illustrated below.

Conjugate B-I with a highly flexible main-chain structure showed higherinhibitory effect on gene expression as compared with conjugate A-I witha less flexible main-chain structure. These results indicate that, inorder to maximize the activity of an antisense nucleic acid inhepatocytes, designing a structurally flexible linker, which is moresusceptible to intracellular metabolism, is important.

The above results indicate that HsPCSK9-1811-LNA(14)-GN(3) issufficiently efficacious even when administered at a dose as low as 0.3to 1 mg/kg once in several weeks and is potentially safer for use intherapy. Also shown is that, for prevention of renal damage, which is amain adverse effect common to antisense oligonucleotide-based drugs, (1)selection of an in vivo highly active antisense oligonucleotide usingthe CEM method, and (2) production of an antisenseoligonucleotide-GalNAc conjugate are effective.

INDUSTRIAL APPLICABILITY

The present invention can be used in the field of pharmaceuticalproducts, in particular, the field of the development and production oftherapeutic agents for diseases associated with a high LDL cholesterollevel.

1. (canceled)
 2. An oligonucleotide conjugate comprising anoligonucleotide and a functional molecule attached to theoligonucleotide via a linker, wherein the functional molecule is capableof altering the activity of the oligonucleotide, wherein the linkercomprises a main-chain linker that binds to the oligonucleotide and aside-chain linker that is branched from the main-chain and binds to thefunctional molecule, wherein the linker has the following structure (I):

wherein the LINKER moiety in the structure (I) has a functional groupcontaining a terminal carbonyl group that forms a covalent bond with theamino group.
 3. The oligonucleotide conjugate according to claim 2,wherein the linker has the following structure (II):


4. The oligonucleotide conjugate according to claim 2, wherein thefunctional molecule is at least one selected from the group consistingof sugars, antibodies, aptamers, intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, thioethers, polyethers,cholesterols, thiocholesterols, cholic-acid moieties, folic acid,lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluorescein, rhodamine, coumarin, and pigments. 5.The oligonucleotide conjugate according to claim 2, wherein thefunctional molecule is at least one selected from the group consistingof sugars, antibodies, and aptamers.
 6. The oligonucleotide conjugateaccording to claim 2, wherein the functional molecule is at least oneselected from the group consisting of cholesterols, thiocholesterols,cholic-acid moieties, folic acid, lipids, phospholipids, biotin, andadamantane.
 7. The oligonucleotide conjugate according to claim 2,wherein the functional molecule is at least one selected from the groupconsisting of polyethylene glycols, thioethers, and polyethers.
 8. Theoligonucleotide conjugate according to claim 2, wherein the functionalmolecule is at least one selected from the group consisting ofintercalators, reporter molecules, polyamines, polyamides,anthraquinone, phenanthridine, acridine, and phenazine.
 9. Theoligonucleotide conjugate according to claim 2, wherein the functionalmolecule is at least one selected from the group consisting offluorescein, rhodamine, coumarin, and pigments.
 10. A method forproducing an oligonucleotide conjugate comprising an oligonucleotide anda functional molecule attached to the oligonucleotide via a linker,wherein the functional molecule is capable of altering the activity ofthe oligonucleotide, wherein the linker comprises a main-chain linkerthat binds to the oligonucleotide and a side-chain linker that isbranched from the main-chain and binds to the functional molecule,wherein the linker has the following structure (I):

wherein the LINKER moiety in the structure (I) has a functional groupcontaining a terminal carbonyl group that forms a covalent bond with theamino group.
 11. The method according to claim 10, wherein the linkerhas the following structure (II):


12. The method according to claim 10, wherein the functional molecule isat least one selected from the group consisting of sugars, antibodies,aptamers, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic-acid moieties, folic acid, lipids,phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluorescein, rhodamine, coumarin, and pigments.